On the first anniversary of the game's shareware version release, an early prototype of the game was made public. It went by the name "LameDuke" and was more in line with Duke Nukem II's story, style, and tone.

At one point in development, the player was able to acquire a space suit which would allow Duke to traverse areas in space without dying.

In the Nintendo 64 version most of the sexual references are gone due to Nintendo being a more family friendly game company. For example, in the cinema behind the blinds for the movie is a ufo, and the porn shop is changed to a comic shop.

In the Duke Caribbean: Life's a Beach expansion, there is a recurring boat named the Kobayashi Maru, named after the Starfleet test of the same name from Star Trek.

In level 1-1 (Hollywood Holocaust), there is a phone number above a urinal. The number, 867-5309, is a reference to the popular song "Jenny" by Tommy Tutone.

In the Mac version of the game, typing in DN1984 on the first level will print "Big Brother is Watching!" at the top of the screen and replace the movie playing in the theatre with the 1984 Super Bowl Apple Ad.

The God-mode cheat "dncornholio" is thought to be a reference to Beavis's alter-ego 'Cornholio' in the TV show Beavis and ButtHead.

In the original versions, the babes kidnapped by the aliens were woven into cocoons and tied up on pillars. Pressing the action button in front of them will make them moan "Kill me...", which is a reference to a similar scene in the Alien franchise. In the Nintendo 64 version, the babes were covered up more (as they were either naked or only had underwear on), and could not be killed (which is how you "saved" them before). The action button now makes the disappear and the message "Babe saved!" is shown.

The box art for Duke Nukem 3D was inspired by the movie poster for Army of Darkness.

In the Stadium level, if players either look up or use a jetpack, they can see a blimp which reads "DUF BEER", a reference to the drink Duff Beer from the television series "The Simpsons".

In level 2-8, Lunar Apocalypse-Lunar Reactor, there is a reference to Star Wars Episode V: The Empire Strikes Back. You can find a dead Luke Skywalker hanging upside down at the back of a cavern. In the N64 version of the game, Luke is replaced with Yoda.

In the level 2-3, Lunar Apocalypse-Warp Factor, there are a few hidden rooms referencing Star Trek: the Next Generation. You can find the Bridge of the Starship Enterprise and Captain Picard's Ready Room, which includes his desk and fish tank. There is also a secret "Really Ready Room" inside Picard's Ready Room, suggesting that Picard had a secret porn stash.

In level 3-10, Shrapnel City-Freeway, there is a hidden Terminator reference. You can find a flattened T-800 type robot at the back of a machine press.

The TV in the bar on the level Red Light District shows a white bronco speeding down the highway. This is a reference to the OJ Simpson car chase.

A reference to "Doom" can be found near the church in the level "Death Row". If you press the panel in front of the cross, the screen will become red tinted and a wall behind the left pane of stained glass will drop down. Following the path will reveal a bloody corpse of the protagonist from Doom. Duke will also comment, "That's one doomed space marine."